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Sample GSM3528720

Query DataSets for GSM3528720

Status

Public on Dec 24, 2018

Title

Capture-C of Hoxa1 in Mus musculus: WT rep1

Sample type

SRA

Source name

embryo, blastocyst embryo, blastocyst

Organism

Mus musculus

Characteristics

genotype/variation: WT
cell type: Embryonic stem cell
strain: 129/Ola
treatment: RA induced differentiation
cell line: E14

Treatment protocol

For RA treatment, ESCs were induced to differentiate by LIF/2i withdrawal and addition of 2uM RA (Sigma). Culture medium was replaced every day.

Growth protocol

Mouse ESCs were grown in culture dishes coated with 0.1% gelatin in Glasgow Minimum Essential Medium (GMEM) supplemented with 15% fetal bovine serum (FBS), 100nM nonessential amino acids, 1% sodium pyruvate, 200mM glutamate, 1% penicillin- streptomycin, 50uM β-mercaptoethanol, 10ng/mL LIF, 1uM PD0325901 (a MEK inhibitor) and 3uM CHIR99021 (a GSK inhibitor). The medium was replaced every 2-3 days (32,33)

Extracted molecule

genomic DNA

Extraction protocol

1. For RNA -seq: cells were lysed with Trizol reagent (Life Technologies) and RNA was extracted according to the manufacturer's instructions. 2. For Capture-C：Briefly, cells were fixed with 1% (vol/vol) formaldehyde for 10 min at room temperature, quenched with 125 mM glycine in PBS, and then lysed in cold lysis buffer (10 mM Tris–HCl, pH7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 0.2% NP-40, 1× complete protease inhibitor cocktail (Roche)). Chromatin was digested with DpnII (New England Biolabs) at 37°C overnight. Fragments were then diluted and ligated with T4 DNA ligase (Takara) at 16°C overnight. Cross-linking was reversed by overnight incubation at 60°C with proteinase K (Bioline). Then 3C libraries were purified by phenol-chloroform followed by chloroform extraction and ethanol-precipitated at −80°C overnight. Sequencing libraries were prepared from 10μg of the 3C library by sonication to an average size of 200 ~300 bp and indexed using NEBnext reagents (New England Biolabs), according to the manufacturer's protocol. Enrichment of 2μg of an indexed library incubated with 3uM of a pool of biotinylated oligonucleotides (probe sequences are listed in Supplementary Table 7) was performed using the SeqCap EZ Hybridization reagent kit (#05634261001, Roche/NimbleGen), following the manufacturer's instructions. Two rounds of capture employing 48~72 and 24 hr hybridizations, respectively, were used. Correct library size was confirmed by agarose gel electrophoresis, and DNA concentrations were determined using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific). All sequencing was performed on Hi-Seq 2500 platforms using paired 150 bp protocols (Illumina).
The Capture-C library was constructed with NEBNext DNA Library Prep Master Mix Set for Illumina (NEB).

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

E14_RA-ed_hoxa1_capture-c_combined_interaction.wig

Data processing

Library strategy: Capture-C
RNA-seq analysis: Clean reads were mapped to Ensemble mm10 using Hisat2 with default parameters. Gene reads were counted by Htseq. Fold changes were computed as the log2 ratio of normalized reads per gene using DEseq2 R package.
Capture-C analysis: The analysis of Capture-C processing step referred to previously reported procedures (Davies, J.O., Telenius, J.M.2016 Nature methods).
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include genes count for each Sample and wig

Submission date

Dec 23, 2018

Last update date

Dec 24, 2018

Contact name

Dianhao Guo

Organization name

Nankai University

Street address

94 Weijin Road

City

Tianjin